Aminopeptidase N (APN/CD13) is a zinc-dependent type II trans-membrane metallopeptidase, belonging to the Gluzincins subfamily of the M1 family, which binds to cellular membrane in a form of a homodimer (Nucleic Acids Res.,1999, 27(1):325-331). CD13 is expressed on diverse cell surfaces, such cells as central nervous system synapse cells, synovial fluid fibroblasts, activated endothelial cells, liver cells, intestinal epithelial cells, placenta, bone marrow progenitors, monocytes, osteoclasts and so on, particularly is enriched on kidney and intestinal brush border (Haema., 2003, 4(6):453-461). Moreover, compared with normal cells, many tumor cells have a relatively high expression of CD13 on their cell surface, such as melanoma, ovarian cancer, prostate cancer, colon cancer, pancreatic cancer, breast cancer, lung cancer and so on.
In recent studies, it is found CD13 is a multifunctional protein, playing a role as proteolytic enzyme, virus receptor, signal transduction molecule on cell surface and so on, while CD13 is involved in cancer invasion, metastasis, and angiogenesis (Current Medicinal Chemistry, 2007, 14, 639-647). Recently, it is reported that the CD13 on surface of tumor cells could change surrounding tissue microenvironment, and has a significant effect on angiogenesis (PNAS 2012, 109, 1637-1642). In addition, Haraguchi etc. suggest that CD13 is a functional bio-marker of semi-dormant liver cancer stem cells (HCSCs) in Human hepatoma cells and its clinical samples, and could prevent an increasing of Reactive Oxygen Species inducing by radiotherapy/chemotherapy to increase resistance for treatment (J Clin Invest 2010, 120, 3326-3339). It is well-known that tumor stem cells are the main factor causing tumor chemotherapy resistance, recurrence and metastasis. It is shown in further studies that, CD13+ liver cancer cells proliferate slowly, and have a capability of self-renewing, differentiating and resisting to treatment, and are related to tumor resistance. CD13 inhibitors and CD13 neutralizing antibody can both induce cell apoptosis and affect tumor cells with multi-drug resistance, cytotoxic anti-tumor drug and CD13 inhibitor can highly enhance inhibiting and killing of tumor, thus the combination of ubenimex with other cytotoxic anti-tumor drugs can highly enhance therapeutic effect compared with ubenimex, what is more, can prevent recurrence and metastasis of tumor (J Clin Invest 2010, 120, 3326-3339).
Ubenimex (Ubenimex, Bestatin, Ube) is a compound with a dipeptide structure found from a nutrient solution of Streptomyces olivoreticuli, which was used as immunoenhancement for the treatment of leukaemia in 1987 in Japan. And then it was on market in 1998 in China with a trade name Bestatin. Bestatin is an immunoenhancement with targeted anti-cancer capacity and dual mechanism. Lots of studies have reported an inhibiting activity of Bestatin on CD13 with IC50 value of 2.5-16.9 μM against CD13. In vitro, Bestatin can inhibit an invasion of a murine melanoma B16BL6 with high metastasis; and also can inhibit a tubular structure formation of HUVECs (Cancer let, 2004, 216(1):35-42). Moreover, mice xenografts experiments suggest that Bestatin can inhibit carcinoma metastasis and angiogenesis induced by tumor cells (Bio. Pharm. Bull., 1996, 19(1):6-10); in clinical study, Bestatin can cooperate with chemotherapy, radiotherapy and combination application to be used in treatment of disease such as leukaemia, multiple myeloma, myelodysplastic syndrome, and other diverse solid tumors. However, mechanism of synergy effect thereof is uncertain.
Recently, in a mice liver cancer xenograft experiment, the combination of ubenimex and 5-Fluorouracil presented an enhanced effect on inhibiting tumor comparing with a single treatment using ubenimex or 5-Fluorouracil (shown in FIG. 1, 2). Otherwise, it was found in further experiment that, the liver cells of a mouse treated by 5-Fluorouracil before was transplanted into other mouse, and by comparing the tumor recurrence of a mice group using ubenimex with the mice group without using ubenimex, it results that, tumor did not recur in the mice group using ubenimex, but the mice group without using ubenimex presented tumor recurrence cases. This study provides great evidence for combination of ubenimex with 5-Fluorouracil and other chemotherapeutics (J Clin Invest 2010, 120, 3326-3339). And it also validates the importance of inhibiting tumor stem cells in tumor chemotherapy.
5-Fluorouracil (5-Fluorouracil, 5-Fu) is a kind of pyrimidine fluoride with a chemical name of 2,4-dihydroxy-5-fluoropyrimidine and belongs to antimetabolite anti-tumor drugs, and can inhibit thymidylate synthase, block deoxythymidylate converting into thymidylate, interfere with DNA synthesis and inhibit RNA synthesis in some degree. 5-Fluorouracil is a widely used clinical anti-tumor drug for treatment of colon cancer, colorectal cancer, stomach cancer, breast cancer, ovarian cancer, choriocarcinoma, malignant mole, head and neck squamous cell carcinoma, skin cancer, liver cancer, bladder cancer, etc., which presents no cross resistance with other general anti-tumor drugs. Otherwise, many antitumor drugs combining with 5-FU exhibit enhanced cytotoxic effect, there has been some studies on drug combination of 5-FU for treating leukaemia (Phytomedicine 18 (2011) 362-365). However, 5-FU is used widely in clinical and has therapeutic effect on many tumor diseases, while having many disadvantages: the effective dose of 5-FU is less different with its toxic dose, which induces the relatively severe myelosuppression and gastrointestinal toxicity in clinical. Short half-life, poor metabolic stability (degraded to FUH2 by dihydropyrimidine dehydrogenase soon) and some tumors with high expression of thymidylate synthase resisting to it are also its disadvantages. Because of oral irregular, large individual differences, low fat-solubility and poor tissue penetration, it is difficult to prepare a convenient oral formation of 5-fluorouracil, therefore, it is administrated by artery or vein mostly, and is not suitable for the long-term chemotherapy.
In decades, many researchers want to reduce or avoid the disadvantages of 5-FU. Among which, the main method is developing pro-drug of 5-FU to improve metabolic stability, reduce toxicity and increase therapeutic index of it. So far, many pro-drug derivatives of 5-FU have been synthesized and some of those have been used in clinial, such as tegafur (FTO), carmofur (HCFU), doxifluridine, capecitabine (Xeloda), atofluding (ATFU), BOF-A2 and so on. Xeloda is one of those approved by FDA to be an oral fluoropyrimidine drug with the best clinical effect in present, which could meet or exceed the efficacy of fluorouracil by continuous intravenous administration; atofluding can be progressively degraded into TFU and 5-Fu in body so as to have continuous effect. In this invention, by using Xeloda and TFU as the positive control drugs, we evaluate the oral pharmacodynamic effect of a multi-target ubenimex-5-FU binded pro-drug derivative synthesized by ourselves.
Hydroxyurea (Hydroxycarbamide), a nucleoside diphosphate reductase inhibitor, can block reduction of nucleotides to deoxynucleotides, interfere with biosynthesis of purine and pyrimidine bases, selectively impede DNA biosynthesis but has no impact on RNA and protein biosynthesis. As a cell cycle-specific drug, cells in s-phase are sensitive to it. Now, it is mainly used for the treatment of solid tumors, such as malignant melanoma, stomach cancer, colon cancer, breast cancer, bladder cancer and so on.
Epirubicin (Epirubicin), an antibiotic anti-tumor drug, is an isomer of doxorubicin, which can directly embed in DNA base pairs in nucleus to interfere with transcription progress, and then prevent formation of mRNA to inhibit biosynthesis of DNA and RNA. Moreover, many other researchers find that epirubicin can inhibit the topoisomerase II and is a cell cycle nonspecific agent. Now, it is mainly used for the treatment of solid tumors in clinical, such as acute leukemia, nephroblastoma, soft tissue sarcoma, bladder cancer, testicular cancer, prostate cancer, stomach cancer, malignant lymphoma, breast cancer, bronchial carcinoma, ovarian cancer, liver cancer (including primary hepatocellular carcinoma and metastatic hepatocellular carcinoma) and so on. So far, combination application of epirubicin has been widely reported, for example, combination of epirubicin with paclitaxel or sorafenib is useful for treatment of advanced breast cancer.
Dasatinib (Dasatinib), explored by the Bristol-Myers Squibb corporation, was approved by FDA in June of 2006 for treatment of imatinib-resistant CML patients or patients with imatinib failure. As a kind of oral tyrosine kinase inhibitor, it can treat chronic myeloid leukemia by preventing leukemia cell excessively proliferating in CML or Ph+ALL patients through targeting tyrosine kinase inhibition enzyme cascade. Other than, Dasatinib can inhibit Src kinase, thus can prevent other human tumor cells with no BCR-ABL expression, such as PC-3 (prostate cancer cells, IC50 is 5˜9 nmol/L), MDA-MB-211 (breast cancer cells, IC50 is 10˜12 nmol/L), and WiDr (colorectal cancer cells, IC50 is 38˜52 nmol/L), etc. (Proc Am Assoc Cancer Res, 2005, 46: 159).
Lenalidomide (Lenalidomide/Revlimid), explored by the U.S. Celgene corporation, is a derivative of thalidomide with a chemical name of 3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione and is used for treating myelodysplastic syndrome and combining with dexamethasone for treating multiple myeloma. Lenalidomide has an impact on multiple intracellular biological pathways. Now, all the phases I, II, III clinical researches have been ended. The studies of phase I and phase II clinical researches demonstrate that it has inhibitory effect on many tumors such as multiple myeloma, prostate cancer, thyroid cancer, renal cancer, melanoma, liver cancer and chronic lymphocytic leukemia (Cancer Chemotherapy and Pharmacology, 2012, 1-14).
The chemical structures of ubenimex, 5-fluorouracil, hydroxyurea, cyclophosphamide, gemcitabine, tirapazamine, lenalidomide, hydroxycamptothecine, epirubicin, dasatinib, and paclitaxel are shown as below:

Multi-targeted drug (multitarget drugs) is an important trend in today's drug research. A multi-targeted drug refers to a drug which can simultaneously act on diverse targets associated with one disease to have a synergistic therapeutic effect, and exhibits better therapeutic effects, less side effects by completely regulating multiple targets associated with one disease, and is especially useful for treating various major diseases associated with complicated pathological mechanism and polygene, including leukemia and other malignancies, central nervous system diseases, cardiovascular diseases and metabolic disorders and so on. So far, multi-targeted treatment using multi-targeted drug is one of the most effective treatments for therapy of various malignancies including leukemia.
A mutual pro-drug or a multi-targeted drug has single physical and chemical property and uniform pharmacokinetic characteristics when comparing with drug combination and a multi-targeted drug may avoid problems such as interaction of different components and differential absorption, distribution and metabolism of different components in a drug mixture, and thus can have synergistic effect, especially in a case of low concentration. A multi-targeted drug shows an obviously better therapeutic effect than combination of corresponding single-targeted drugs. Therefore, it is of great significance to design and synthesize a novel anti-cancer drug with synergistic therapeutic effect based on hybridization principle to be used to treat tumor recurrence, metastasis and drug resistance, especially binding ubenimex as a tumor stem cells inhibitor with other anti-tumor drugs.